The insolubility of the scrapie prion protein (PrPSc) has frustrated all attempts to solve its structure by X-ray crystallography or NMR spectroscopy. Recently, we reported the discovery of two-dimensional (2D) crystals of the N-terminally truncated PrPSc (PrP 27-30) and a redacted miniprion (PrPSc106). Analyzing the 2D crystals by electron crystallography allowed us to map the differences between PrP 27-30 and PrpSc106. These data were used to constrain structural models of PrPSc. We propose to investigate the parameters that govern growth of the 2D crystals in order to obtain specimens suitable for low dose, cryo-electron crystallography. These crystals will be used to collect higher resolution data to generate a three-dimensional reconstruction of the structure of PrPSc. We will also pursue various labeling techniques to localize different parts of the molecule in order to position computational models of PrPSc on the crystal lattice. The experimental data will be used to refine structural models of PrPSc that will be based on structures of known proteins or domains of proteins. In particular, we will focus on the parallel Beta-helix as a motif that can account for the secondary structure constraints implied by FTIR spectroscopy data and spatial constraints determined by electron microscopy. While fiber diffraction results have historically contributed only low-resolution structural data to our analysis of the structure of PrPSc, we believe that when coupled with the models and electron crystallography data, we will be able to extract more meaningful information out of these studies. Therefore, we want to revisit fiber diffraction studies using more advanced methods of fiber alignment coupled with synchrotron-based X-ray and electron diffraction approaches. Furthermore, we want to explore which of the well-established prion strains are most suitable for structural analysis by either electron crystallography or by fiber diffraction. We are also interested in studying PrP sequence truncations that can support PrPSc formation in transgenic animals. This work will include PrP 89-231, a homolog of PrP 27 - 30, and variants of PrP106 that extend or shrink the internal deletion from 141 - 175 yet still support PrPSc formation.